Abstract

1994

Interactions between the novel benzamide histone deacetylase inhibitor (HDAC inhibitor) MS-275 and fludarabine were examined in lymphoid and myeloid human leukemia cells in relation to mitochondrial injury, signal transduction events, and apoptosis. Prior exposure of Jurkat lymphoblastic leukemia cells to a marginally toxic concentration of MS-275 (e.g., 500 nM) for 24 hr sharply increased mitochondrial injury, caspase activation, and apoptosis in response to a minimally toxic concentration of fludarabine (500 nM), resulting in highly synergistic antileukemic interactions and loss of clonogenic survival. Simultaneous exposure to MS-275 and fludarabine also led to synergistic effects, but these were not as pronounced as observed with sequential treatment. Similar interactions were noted in the case of a) other human leukemia cell lines (e.g., U937, CCRF-CEM); b) other HDAC inhibitors (e.g., sodium butyrate, SAHA); and c) other nucleoside analogs (e.g., ara-C, gemcitabine). Potentiation of fludarabine lethality by MS-275 was associated with acetylation of histones H3 and H4, down-regulation of the anti-apoptotic proteins XIAP and Mcl-1, enhanced cytosolic release of pro-apoptotic mitochondrial proteins (e.g., cytochrome c, Smac/DIABLO, and AIF), and caspase activation. It was also accompanied by the caspase-dependent down-regulation of p27KIP1, cyclins A, E, and D1, and cleavage and diminished phosphorylation of pRb. However, increased lethality of the combination was not associated with enhanced fludarabine triphosphate formation or DNA incorporation, and occurred despite a slight reduction in the S-phase fraction. Prior-exposure to MS-275 attenuated fludarabine-mediated activation of MEK1/2, ERK, and Akt, and enhanced JNK phosphorylation; furthermore, inducible expression of constitutively active MEK1/2 or Akt significantly diminished MS-275/fludarabine-induced lethality. Combined exposure of cells to MS-275 and fludarabine was associated with a significant increase in generation of reactive oxygen species (ROS); moreover, both the increase in ROS and apoptosis were largely attenuated by co-administration of the free radical scavenger L-N-acetylcysteine (LNAC). Finally, prior administration of MS-275 markedly potentiated fludarabine-mediated generation of the pro-apoptotic lipid second messenger ceramide. Together, these findings indicate that the HDAC inhibitor MS-275 induces multiple perturbations in signal transduction, survival, and cell cycle regulatory pathways that lower the threshold for fludarabine-mediated mitochondrial injury and apoptosis in human leukemia cells. They also provide insights into possible mechanisms by which novel, clinically relevant HDAC inhibitors might be used to enhance the antileukemic activity of established nucleoside analogs such as fludarabine.